Study On The Molecular Mechanisms Of The Effects Of Curcumin On The Growth And Proliferation Of HT-29 Cells

Curcumin is an important polyphenol extracted from the rhizomes of Curcuma longa L.Several studies have shown curcumin exerts antioxidant,anti-inflammatory, anti-carcinogenic,Alzheimer's preventional and antiarthritic activities.This study will focus on the molecular mechanisms of the effects of curcumin on the growth and proliferation of HT-29 cells.In this study,we observed treatment with 10-80μM curucmin inhibited the growth of HT-29 cells by microscopic wounding assay.Hoechst 33258 staining showed that curcumin significantly induced apoptosis of HT-29 cells.Exposure to 10-80μM curucmin also markedly induced the mitochondrial membrane potential(ΔΨm) collapse and activation of caspase-3(P＜0.05).To investigate the mechanisms of curcumin induced apoptosis of HT-29 cells,the release of cytochrome c from the mitochondria and the apoptosis-related proteins such as Bax,Bcl-2,Bcl-xL,Bad,caspase-3,PARP,and survivin were determined by western blotting analysis and their mRNA expressions were assayed by reverse transcriptase-polymerase chain reaction(RT-PCR).A significant decrease in expression of the anti-apoptotic protein such as Bcl-2,Bcl-xL and survivin was observed after exposure to 10-80μM curcumin(P＜0.05),while the levels of pro-apoptotic protein such as Bax and Bad increased in the curcumin-treated cells (P＜0.05).Curcumin also induced the release of cytochrome c,the activation of caspase-3,and the cleavage of PARP in a dose-dependent manner(P＜0.05).These data suggested that curcumin induced the HT-29 cell apoptosis possibly was via the mitochondria mediated pathway.Irregular activation ofβ-catenin/Tcf-4 signaling pathway occurs in many colorectal cancer cells.We studied the effects of curcumin on the level ofβ-catenin in HT-29 cells by western-blotting.The results showed that incubation with 20-80μM curcumin could induce cleavage ofβ-catenin.The level ofβ-catenin/Tcf-4 complex in HT-29 cell nucleus was studied by co-immunoprecipitation and western blotting.The expression of the target genes such as c-myc and cyclinD1 was investigated by RT-PCR and western blotting.The results showed that the association ofβ-catenin with Tcf-4 in nucleus could be inhibited by curcumin(P＜0.05).The expression of c-myc and cyclinD1 was also downregulated by curcumin.Pretreatement of HT-29 cells with Z-DEVD-FMK,a specific inhibitor of caspase-3,could inhibit the curcumin induced cleavage ofβ-catenin.However,curcumin induced downregulation of c-myc and cyclinD1 could not be blocked by Z-DEVD-FMK.These results suggested curcumin could induce the cleavage ofβ-catenin by activition of caspases and downregulate the activity ofβ-catenin/Tcf signaling pathway independent of the caspases in HT-29 cells.We also studied the expression of the target genes ofβ-catenin/Tcf-4 signaling pathway such as PPARδ,VEGF,COX-2 and 14-3-3εin HT-29 cells.The results showed that curcumin treatment decreased the expression of PPARδ,VEGF,COX-2 and 14-3-3ε(P＜0.05).In cytoplasm,14-3-3εcan associate with the pro-apoptotic protein,Bad,and inhibit the pro-apoptotic activity of Bad.Our results showed curcumin might increase the level of Bad by down-regulation of 14-3-3εin HT-29 cells.To investigate the molecular mechanism of curcumin induced HT-29 cell death and growth inhibiton,we studied the anti-oxidant and pro-oxidant activities of curcumin. The in vitro assay showed curcumin had an effective DPPH^·scavenging,ABTS⁺ scavenging,superoxide anion radical scavenging,hydroxyl free radical scavenging, ferric ions(Fe³⁺) reducing power activities.Curcumin could also inhibit the peroxidation of linoleic acid and the oxidative damage of pBR322 DNA induced by hydroxyl free radical.The in vivo assay showed curcumin could increase the levels of the reactive oxygen species(ROS) and glutathion in HT-29 cells(P＜0.05). N-acetylcysteine(NAC) and catalase pretreatment could significantly inhibit curucmin induced increase of ROS and caspase-3 activation(P＜0.05),which indicated curcumin indcued HT-29 cell apoptosis might be mediated by the stimulated increase of ROS.